PROJECT SUMMARY Protein synthesis is tightly controlled by translation initiation factors that fine-tune gene expression to accommodate changes in the cellular environment. The long-term goal of our research is to understand the molecular regulation of eukaryotic translation initiation factors, especially eIF6. Among the eukaryotic initiation factors, eIF6 is unique as it plays a dual role in controlling the initiation of translation and in aiding the biogenesis of the 60S ribosomal subunit. eIF6 directly binds to the 60S subunits and sterically hinders associations between the 60S and 40S ribosomal subunits. Release of eIF6 from the 60S subunits allows 60S to associate with the mRNA-loaded 40S subunits to form translationally competent 80S complexes, which initiates translation. eIF6 role in translation is indispensable, therefore its levels and interaction with the 60S must be stringently regulated to ensure successful commencement of protein synthesis. Translational control is achieved by modulating eIF6 levels, phosphorylation status, and activity in response to a slew of growth and stress signals. eIF6 regulation is especially significant for cancer as disruption of its activity restricts tumorigenesis. eIF6 is overexpressed in several cancers and its enhanced expression is associated with advanced stages of cancers that have a poor prognosis. While much is known about the physiological and pathological states that alter eIF6 expression, there is a huge gap in our understanding of the mechanisms that regulate eIF6 under those conditions. Towards addressing this gap in knowledge, our work has identified a novel phosphorylation-dependent mode of regulation and we propose to elucidate its functional relevance (aim1) and examine the dynamics of eIF6 and 60S interactions and decipher its role in tumorigenesis (aim 2). Our work will use mammalian cell lines and a rigorous set of cellular, molecular, and biochemical tools to address eIF6 regulatory mechanisms, and will provide information for tailoring therapeutics focused on eIF6 and its regulators.